The development of vaccine to combat Enterotoxigenic E. coli (ETEC) is vital given the pathogen's global impact on 200 million individuals annually; however, an entirely new, non-conventional approach is needed. Field studies have suggested that inert, antigen-based vaccines to combat ETEC have essentially no efficacy. Therefore, a vaccine that mimics a natural infection (i.e. a live-attenuated, but not knockout strain) will elicit the most robust, protective response. Building on a recently successfu advance developed by the principal investigator that uses synthetic biology to construct bacterial vaccines, gene design software will be used to 're-code' the ETEC heat-labile (LT) and heat-stable (ST) toxins to have significantly decreased, but not eliminated, levels of expression. This 're-coded' strain could in the future serve as a live-attenuated vaccine capable of immunizing against ETEC, the most common cause of diarrhea in the developing world. This approach developed by the PI is expected to yield positive results in ETEC because the approach, specifically toxin customization, has been successfully utilized for the development of a novel, live-attenuated Streptococcus Pneumoniae (SP) vaccine candidate (Coleman JR et al. JID 2011). Synthetic gene customization will achieve the construction of a live-attenuated vaccine-strain capable of immunizing against ETEC by swapping the genes encoding the target toxin with synthetic genes that have been 're-coded' to use codon-pairs that slow the rate of translation. The synthetic re-design will result in decreased, but not entirely eliminated, toxin expression (Coleman JR, Science 2008). Previous findings using SP strains expressing low levels of synthetic virulence factors were significantly less virulent than the wild type, yet coul induce a protective immune response. The PI found that synthetic bacterial strains expressing non-damaging levels of toxin were even less virulent than a control knockout strain, suggesting very low levels of virulence factor production may be needed to induce immunity and we believe this finding is pertinent for ETEC vaccine construction (Coleman JR, JID 2011). We believe a similar finding will occur in synthetically modified ETEC strains. The heat-labile (LT) toxin of ETEC is required for attachment and colonization on the intestinal mucosa; thus all killed and LT knockout strains in the past have been ineffective as vaccines (Johnson AM, J Bac. 2009; Qadri, F. Vaccine 2004). Furthermore, it is known that protection against a second natural infection was attributed mostly to the LT and ST toxin profile of the naturally infecting strain an much less to the specific serotype of the strain (Johnson AM, et al. 2009). Therefore, by utilizing SAVE 're-coding' of the LT and ST toxin genes, one could construct vaccine strains that secrete low-levels of the wild type toxins, which are a known necessity for subsequent immunity. We hypothesize that synthetically modified ETEC producing low, sub-pathogenic levels of these toxins will provide the necessary host attachment and induction of the immune response as seen in SP. The SAVE approach could provide the solution to what has been sought by the ETEC vaccine field for decades - inclusion of the LT and ST toxins at levels capable of immune stimulation however low enough to avoid toxicity (diarrhea) (Steinsland H. J Clin Microbiol 2004). We have performed an initial pilot experiment and have transformed the ETEC H10407 strain with one derivation of a 'de-optimized' LT toxin and seek funds here to support the construction of additional strains and characterize them in vitro and in vivo. This project is ideally suited for the STTR program - a defined experiment in Phase I, which if successful represents a significant advance and will allow for expansion in Phase II. At the conclusion of this Phase I, we believe we will have a well-characterized live-attenuated vaccine strain to combat ETEC. In Phase II this strain will be further characterized in vivo in an animal system that actually mimics human infections (piglets) and possibly in the clinic. Results from this study could have a very large global impact given that currently there is no vaccine against ETEC. We seek to simultaneously yield high impact results while expanding the applicability of rational gene design to bacterial pathogens.